1. Field of the Invention
The present invention relates to fluorescence detection and more particularly to the collection of fluorescent emission from a capillary separation channel.
2. Description of Related Art
Fluorescence detection is inherently very sensitive and macromolecules (e.g. proteins and DNA fragments) of a biological sample which have been labeled with fluorescent materials can be detected by analyzing the fluorescent emissions. Fluorescence detection has been practiced in the field of capillary electrophoresis. Reference is made to U.S. Pat. No. 4,675,300 to Zare et al for a detailed description of the detection technique. In general, Zare makes use of a coherent light source (e.g. laser) to interrogate a small portion of a capillary separation channel thereby defining a small detection volume. The samples being separated by electrophoresis have been tagged with fluorescent materials such as fluorescein prior to, during or just after electrophoresis. Upon electrophoresis, as the samples cross the path of the light beam, the fluorescent materials are caused to fluoresce thereby indicating the presence of a sample. By tagging the samples with appropriate fluorescent materials, one can detect the intensity of fluorescent emission from the separated samples and determine from the relative intensities the amount of a particular sample present in the sample mixture and its identity.
To date, laser-induced fluorescence is by far the most sensitive means of detecting many types of sample components separated by capillary electrophoresis. Because of the small detection volume, it is desirable to collect as much of the fluorescent emissions as possible to achieve maximum sensitivity. In the past, carefully corrected optics such as a large numerical aperture microscope objective or one or more optical fibers have been used to collect a relatively small solid angle of the fluorescent emission. In this case not only must the laser beam be in proper alignment with the capillary separation channel, but the capillary separation channel itself must be brought into proper alignment with the microscope objective. This necessitates the use of a translatable optical alignment component such as a capillary positioner or a microscope objective positioner. Since careful alignment of both the capillary separation channel with the laser beam and the collection optic (microscope objective or optical fibers) are essential in order to realize maximum sensitivity, typically at least two of the optical components have been mounted on translatable platforms in order that the system may be brought into optimum alignment.
The design of the collection optic system must also take into consideration the undesirable scattered radiation at the source frequency which is typically much more intense than the fluorescent emission. It is known that there is a bright plane of scattered radiation at the source frequency which is distributed over a 360.degree. angle about and perpendicular to the capillary column. Because it is necessary to minimize the amount of scattered radiation reaching the photodetector, in the past some optical systems have been configured to collect emissions which are out of this plane of scatter.